This invention relates to the automated processing and separation of biological cells as found in whole blood, and relates more specifically to a functionally closed system allowing to extract certain cell populations like hematopoietic stem cells, for immediate use or their mixing with an additive solution or a storage solution for later separate storage operations and to the methods for carrying out such an extraction.
Blood separation systems and methods have emerged over the past 20 years in response to the growing need for efficient blood component therapies. Among them are the transplantation of hematopoietic progenitor stem cells, which in many cases is the only remaining cure to oncological disorders. Patients in need of a stem cell transplant have mainly three options:
1) Adult bone marrow stem cells;
2) Peripheral blood stem cells found in the circulatory system;
3) Stem cells found in umbilical cord and placental blood retrieved at birth of a new born infant.
For most stem cell transplants, the main limitation has been the risk of graft-versus-host-disease (GVHD), requiring an excellent HLA-match tissue (HLA=Human Leucyte Associated).
Umbilical cord blood is a rich source of the primitive hematopoietic stem and progenitor cells, with extensive proliferation capacity and capacity to self-renew. This field has advanced rapidly from clinical implants utilizing only HLA-matched grafts to unrelated donor cell transplants which open a much larger indication for stem cell transplantation. This increase in clinical experience with cord blood is due mainly to the establishment of banks for storage of hematopoietic stem cells from unrelated umbilical blood cord.
Blood volumes recovered from umbilical cord are usually very low (40 to 150 ml) and there is some concern that any attempt at product manipulation and concentration might result in stem cell loss, which might impair engraftments. Therefore umbilical cord blood is sometimes stored as is, with preservative solution added. A much preferred way would be to eliminate most unwanted cells like red cells and white cells, resulting in a considerable volume reduction. Less preservative solution would be required, smaller bags, smaller storage spaces would be used and considerable energy savings achieved, all of this translating in substantial cost savings. The quality of the stem cell product when retransfused would be improved as well, as lysed cells resulting from storage would be drastically reduced.
No device or automated system exists for processing and concentrating on line umbilical cord stem cells. There is nevertheless a considerable interest for concentrating umbilical cord blood stem cells without loss or altering their functionality.
EP-B-0 912 250 (C. FELL), the contents whereof are herein incorporated by way of reference, describes a system for the processing and separation of biological fluids into components, comprising a set of containers for receiving the biological fluid to be separated and the separated components, and optionally one or more additional containers for additive solutions. A hollow centrifuge processing chamber is rotatable about an axis of rotation by engagement of the processing chamber with a rotary drive unit. The processing chamber has an axial inlet/outlet for biological fluid to be processed and for processed components of the biological fluid. This inlet/outlet leads into a separation space of variable volume wherein the entire centrifugal processing of biological fluid takes place. The processing chamber comprises a generally cylindrical wall extending from an end wall of the processing chamber, this generally cylindrical wall defining therein the hollow processing chamber which occupies a hollow open cylindrical space coaxial with the axis of rotation, the axial inlet/outlet being provided in said end wall coaxial with the generally cylindrical wall to open into the hollow processing chamber. The processing chamber contains within the generally cylindrical wall an axially movable member such as a piston. The separation space of variable volume is defined in an upper part of the processing chamber by the generally cylindrical wall and by the axially movable member contained in the generally cylindrical wall of the processing chamber, wherein axial movement of the movable member varies the volume of the separation space, the movable member being axially movable within the processing chamber to intake a selected quantity of biological fluid to be processed into the separation space via the inlet before or during centrifugal processing and to express processed biological fluid components from the separation space via the outlet during or after centrifugal processing. Means are provided for monitoring the position of the movable member to thereby control the amount of intaken biological fluid and the expression of separated components. The system further comprises a distribution valve arrangement for establishing selective communication between the processing chamber and selected containers or for placing the processing chamber and containers out of communication.
The system according to EP-B-0 912 250 is designed to operate for the separation of biological fluids, and has proven to be very polyvalent for many separation applications, especially for on-line separation of components from a donor or a patient.
According to the invention, such system is arranged to operate in a separation mode and in a non-separation transfer mode, which provides greater possibilities for use of the system including new applications which were heretofore not contemplated, such as separation of hematopoietic stem cells and in general laboratory processing. According to the invention, the system is arranged to operate such that:
in the separation mode fluids can be intaken into the processing chamber while the chamber is rotating or stationary, fluid intaken into the chamber is centrifuged and separated into components, and the separated components expressed while the chamber is rotating or, optionally, for the last separated component, while the chamber is stationary; and
in the transfer mode the processing chamber intakes fluid and expresses fluid with the chamber stationary, The valve actuation arrangement is actuable to transfer amounts of fluid from one container to another via the processing chamber, by moving he member, without centrifugation or separation of the fluid into components, and the means for monitoring the position of the movable member controls the amounts of non-separated fluids transferred.
Further features of the invention are set out in the claims. This invention thus proposes a functionally closed processing kit associated with a portable apparatus, whose function is to monitor and automate the procedure. The kit, usually disposable for avoiding the likelihood of disease transmission, is based on a centrifugal processing chamber whose volume can be varied during operation, allowing to adjust to the exact quantity of blood to process. Such variable volume chamber is described in the aforementioned EP-B-0 912 250 (C. FELL). The chamber is connected to a set of bags and tubing lines for the collection of the separated components. The blood bag containing the blood to process is generally connected to the disposable set through the use of a sterile connecting device, or an aseptic connection under laminar flow. It is however possible to have this bag prefilled with anticoagulant and preconnected to the disposable kit.
A bag containing an additive solution can be connected to the disposable kit via a bacterial filter. The other bags are provided for the collection of the separated components. The stem cell collection bag material is optimally chosen for the storage conditions.
The tubing line selection for conveying the separated products into the proper bags is accomplished by a set of rotational valves called stopcocks that can be arranged in a manifold array, or by a single multiport rotational valve, forming part of the set. Such an arrangement allows to eliminate any cross-contamination between adjacent lines when using standard pinch valves.
The above-mentioned disposable kit cooperates with an instrumentation for monitoring and automating the process, for instance as described in EP-B-0 912 250 (C. FELL). The centrifuge drives a rotating disk which receives the centrifugal processing chamber and locks it in place. Its closing cover will grip and hold she housing of the rotary seal of the processing chamber.
An optical sensor made of an array of LED and corresponding receiving sensors placed at 180xc2x0 is implemented vertically on the side of centrifuge, for monitoring the piston position. Volumes intaken into or extracted from the chamber can therefore be exactly measured. The topdeck receives an optical line sensor module which monitors the color in the effluent tubing, feeding-back this information to the control program. An array of shafts equipped with fittings for driving a set of multiple stopcock valves protrude from the top deck. They are coupled to a set of motors enabling the tubing line selection. Encoders are attached to the motors for monitoring the stopcock valves position. The front panel incorporates a window allowing the user to see displacement of the piston in the chamber.
The procedure for extracting stem cells out of an umbilical cord is as follows. Initially blood is recovered from the umbilical cord at birth and collected aseptically into a plastic bag, with anticoagulant added like Citrate-Phosphate-Dextrose CPD-1 to avoid clotting. After initial sampling is taken to assess its richness in stem cells, the bag is sterile or aseptically connected to the processing kit and the whole set is loaded onto the separation system, which initially operates in the separation mode or the transfer mode at choice. In the separation mode the centrifuge starts driving the separation chamber at around 4000 rpm, and blood is introduced by moving down the chamber piston pneumatically. Two cases can then occur. If the volume of blood to process is smaller than the processing chamber volume (as detected by the empty state of the effluent tubing), the piston is maintained at an intermediate position pneumatically, monitored by the piston position sensor. If the volume of blood completely fills the separation chamber, detected by the piston reaching the bottom of the chamber, the pneumatic compressor stops. In both cases, centrifugation speed is increased to around 6000 rpm to shorten the sedimentation time to 5-8 min. After this period, centrifugation slowly decreases to around 4000 rpm. Stopcocks are rotated to allow the collection of the separated products, and the pneumatic pressure gradually increases to move the piston upwards. The speed of the piston remains low, actively monitored by she piston position sensor, in order to maintain the sedimentation profile of the cells within the chamber. The first milliliters from the inlet line are purged in the stem cell bags. Plasma is extracted then collected into a first bag. It is followed by platelets, packed in the intermediate layers, or buffy-coat. Apparition of the first platelets is detected by the optical line sensor monitoring the effluent line tubing. At that moment, the product, very rich in stem cells, is directed into a second collection bag by rotating a stopcock valve. A volume counter is started, which depends among other factors on the total blood volume processed. When this counting volume has been reached, centrifugation is stopped. The proper stopcock valve is rotated and the last product is extracted, essentially a volume of packed red cells with residual granulocytes, into a third collection bag. Another cycle can then resume if the umbilical cord blood has not been totally processed. Otherwise, the separation and stem cells collection process is completed at this stage.
However, it is possible to reprocess the content of the bag containing the stem cells, in view of further purifying the product. In this case, the proper stopcock valve is rotated to intake the content of the stem cell concentrate bag. The procedure to collect stem cell rich layer is identical then to the one described above.
Another alternative to isolate the stem cell rich fraction from the buffy-coat is by using density gradient products such as those available under the names Ficoll and Percoll. In this alternative, a density gradient product is first introduced into the processing chamber, followed by introduction of whole blood, and a component of the biological fluid is separated into a giver container and its collection is completed when the density gradient appears. Possibly the density gradient product may be introduced during processing.
Using Ficoll would for example consist of first introducing the density gradient into the processing chamber, followed then by whole blood. After complete introduction of blood into the chamber, a sedimentation period of a few minutes is started. Stem cells and platelets form an interface in front of the gradient, whereas erythrocytes and granulocytes have passed through the Ficoll and are held against the walls of the separation chamber. The piston is then lifted gently as in the standard procedure, the stem cell fraction being collected at the apparition of the first platelets. The effluent line clears up again when Ficoll exits the chamber, which is the appropriate moment to stop the collection.
When the stem cells are collected by one of the methods described above adequate preservative solution can be introduced into the processing chamber by rotating the proper stopcock, the system operating in the transfer mode. It is then retransferred into the stem cell bag, its volume accurately controlled by the piston position sensor.
The bag containing the stem cell rich product can be disconnected at this stage from the rest of the set. Its volume ranges between 20-40 ml, depending of the initial volume processed. The by-products of the separation, plasma and packed red cells, can then be used for serology and HLA typing, avoiding any product loss due to sampling in the stem cell bag.
This separation system and method offer significant advantages over manual processing techniques. The disposable kit is a functionally closed system, avoiding any risk of contaminating the product during manipulation. The protocol is fully automated, through a microprocessor based control system, with ability to vary the main parameters, like centrifugation speed, centrifugation time, speed of introduction and extraction, volume to collect, etc. The volume reduction for the stem cell product represents a gain of 50% at least compared with the current state of the art. The instrumentation is very compact and portable, ideal for the decentralized processing of such procedures.
A further aspect of the invention is the use of the above-described system for processing variable volumes of biological fluid from 10 ml up to the maximum volume of the separation chamber, and for adding an additive solution to the separated components, in particular for separation of stem cells from blood and mixing the separated stem cells with a preservative solution; for separation of hematopoietic stem cells from umbilical cord blood; for separation of hematopoietic stem cells from an apheresis collection; and for separation of hematopoietic stem cells from a bone marrow aspirate.